Flask-shaped invaginations of the plasma membrane known as caveolae are important regulators of the cardiovascular and pulmonary systems. The membrane protein caveolin-1 (Cav1) is a major structural component of caveolae and is required for their formation in non-muscle cells. Cav1 is highly expressed in the lung, and has been linked to lung cancer, asthma, pulmonary fibrosis, pulmonary arterial hypertension, chronic inflammatory respiratory diseases, and acute lung injury. Cav1 is also known to regulate vascular homeostasis and was recently identified as one of 15 key drivers of cardiovascular disease. However, the exact mechanisms by which caveolae form and function normally, and how defects in caveolae give rise to disease remain incompletely understood. One of the greatest impediments to our understanding of how caveolae assemble and function is our limited knowledge about how Cav1 is packed within caveolae. The goal of this proposal is to address this major gap in knowledge by defining the atomic-level structure of Cav1 oligomers that are known to serve as the fundamental building blocks of caveolae. To do so, we will utilize a combination of biochemical and spectroscopic approaches, single molecule electron microscopy, cell biological assays, and computational modeling to study purified Cav1 oligomeric complexes. One specific aim is to define the overall architecture of Cav1 oligomers and identify key determinants of their structure and stoichiometry. Another aim is to map the three-dimensional organization of Cav1 within these oligomers and define structural changes in the protein associated with the monomer to oligomer transition. The results of these studies will enable us to answer a number of long-standing questions in the field, including how Cav1 monomers oligomerize to form complexes, what structural features of Cav1 are required for the protein to bend membranes, how Cav1 complexes interact amongst themselves to build caveolae, and what regions of Cav1 are available to bind other proteins and lipids. These insights will be key to furthering our understanding of how Cav1 and caveolae regulate cellular functions that are critical to ensure proper function of the cardiovascular and pulmonary systems. Finally, our studies will also have important implications for personalized medicine by providing a structural framework for understanding how both common variants and disease-associated mutations of Cav1 impact the structure and function of caveolae. !